PROJECT SUMMARY Wnt signaling is used to maintain the regenerative potential of somatic and embryonic stem cells. However, this activation comes at a risk, since ectopic Wnt signaling is highly oncogenic for epithelial tissues; thus, tissues maintained and developed in the presence of ectopic Wnt signaling may have an excess risk of tumor development later in life. We have found an auxiliary function for one of the Wnt signaling receptors, Lrp5, that we propose could be responsible for the oncogenic activity of Wnt signaling; if we are correct, Lrp5 activity could be manipulated in future applications of stem cell-based cures. Study of mammary epithelial stem cells offers a remarkable opportunity to study somatic stem cell activity. Just a single mammary stem cell can regenerate a complete mammary ductal tree upon transplantation to a new host fat pad. Our previous work has shown that 1) mammary stem cell function is directly related to Wnt signaling activity, and 2) the Wnt signaling receptor Lrp5 is required to maintain mammary stem cell activity, despite the fact that the more potent Wnt signaling receptor, Lrp6, is co-expressed. This reflects the fact that the Wnt ligand(s) that maintain mammary stem cells require both Lrp5 and Lrp6 to be present to generate a Wnt ligand. We therefore call Lrp5 a ?gatekeeper? of mammary stem cell function. However, recently we have found 1) that Lrp5 has a profound effect on mammary epithelial cell growth that is independent of the canonical Wnt signaling pathway (?catenin/TCF-dependent transactivation). Lrp5 promotes glucose uptake by a totally novel mechanism, and glucose deprivation is sufficient to phenocopy loss of function of Lrp5; 2) that the unusual soluble Wnt ligand, Wnt3a, can promote growth and development of mammary epithelial cells in vivo, and does not require Lrp5 to be present. Our hypothesis is that Lrp5 promotes tumor development in response to ectopic Wnt signaling, but is not required for the Wnt-induced stem cell accumulation that accompanies the activation of breast regenerative potential. In this application we aim to find out 1) how Lrp5 regulates glucose uptake and mammary epithelial cell growth, and 2) whether Lrp5-mediated glucose uptake is required for the massive increase in glucose uptake that accompanies cell transformation. Thus, increased glucose uptake is required to fuel the metabolic reprogramming characteristic of tumor cells (the ?Warburg? effect), making tumor cells particularly susceptible to deficiencies of glucose uptake. This project aims to 1) define a novel activity for Lrp5, a cell surface receptor previously described as a canonical Wnt signaling receptor; 2) address a novel paradigm, that the oncogenicity of Wnt signaling may be ameliorated by reducing the metabolic activity associated with Lrp5; 3) apply unique technical (IFAST immunoprecipitation) and knowledge resources from the study of Lrp5 in bone (where Lrp5 governs bone density, and mutations of Lrp5 in human patients generate gain- and loss-of-function for osteoblast activity).